An environment containment system provides an adjustably controllable environment for cells entrained within the fluid stream generated by a flow cytometer.
A flow cytometer or cell sorter is an instrument that physically separates cells according to certain parameters. Many cell sorters use techniques to distinguish subpopulations of cells by employing a unique blend of modern technologies such as fluidics, electric fields, lasers, optics, analog and digital electronics, computers and software.
Referring to FIG. 1, in conventional flow cytometry (1), selected cells can be labeled with fluorescent molecules that bind specifically to the constituent(s) (e.g. using a fluorescently-labelled antibody to a particular surface antigen) to be measured. Certain, desired cells (a subpopulation of cells) may, for example, be fluorescently labeled while other cells in the sample of cells are not fluorescently labeled. In this example, such fluorescently labeled cells may be selectively identified from a heterogeneous cell population.
The cells and a sheath fluid are supplied to the interior volume of a droplet generator (2) from a cell source (3) through an injector tube (4) and a sheath fluid source (5) respectively. The sheath fluid confines, by hydrodynamic focusing, the sample of cells to a central core of the laminar flow that leaves the droplet generator (2) at an orifice (6) as a fluid stream (7). The fluid stream entraining the cells to be sorted, pass such cells, typically one by one, through a beam of excitation light from a light source (8) (which can be a laser or arc lamp), such that each fluorescently labeled cell emits a short flash of fluorescence. The intensity of the fluorescence may be proportional to amount of the fluorescent label on the individual cells. These flashes of fluorescence are collected by a fluorescence collection lens, which focuses the light on a sensitive fluorescence detector (9). The fluorescence detector (9) transforms the flashes of light into electrical pulses, which are measured and recorded by an analysis means (10), which may in part include a computer. Thus, multiple parameters including fluorescence at different wavelengths, as well as size and shape or structure, may be analyzed for each individual cell in the sample of cells.
Once the analysis of an individual cell is made, the flow cytometer (1) has the ability to selectively remove certain cells from the fluid stream (7). Droplets (11) are generated in the fluid stream (7) by the oscillation of the droplet generator (2), which is coupled to an oscillation generator (12) and an oscillation frequency controller (13). A droplet-separation point or break off point, which occurs shortly after the cells leave the droplet generator (2) through orifice (6) become selectively charged by a charging pulse. The charging pulse may be produced by a charging system control logic that is coupled to receive signals from the analysis means (10). The charging system control logic analyzes signals from the analysis means (10) and determines whether the cell is to become a selected cell and, if so, applies the charging pulse to the conductive fluid carrying the cells just before the droplet carrying the cell breaks off from the fluid stream (7). The droplet (11) carrying the selected cell separates as a charged droplet from the fluid stream (7) and falls, under the force of gravity, through a constant electric field produced by the deflecting system (14). If the cell is not a selected cell, then charging system control logic will not apply the charging pulse, and the droplet (11) containing this cell will separate from the fluid steam (7) as an uncharged droplet and will fall through the electric field largely unaffected by this field. Typically, one droplet (11) will contain only one cell so that the selective charging of a droplet will select only one cell.
The deflecting system (14) alters the trajectory in which the charged droplets (11) are traveling such that the charged droplets are physically separated from the uncharged droplets according to the value of the parameters analyzed or measured by the analysis means (10). After passing through the deflecting system (14), the droplets are collected in a cell collector (15) that may have several different collection receptacles or containers. Additional background information may be obtained by referring to Flow Cytometry and Sorting, Second Edition by Myron R. Melamed, Tore Lindmo, Mortimer Mendelsohn, published by Wiley-Liss New York, N.Y., 1990. Also see, for example, U.S. Pat. Nos. 5,150,313, and 3,560,754, each hereby incorporated by reference.
Although technology has led the way for a new generation of cell sorters that are simpler to operate and maintain, and significantly less expensive both to purchase and to operate, the inability to analyze, measure, or sort cells in an adjustably controllable environment significantly limits the types of applications to which flow cytometers or cell sorters may be used. And while various solutions have been suggested or proposed such as those disclosed by U.S. Pat. Nos. 5,776,781; 5,641,457; 5,083,558; and 5,200,616, each hereby incorporated by reference, significant problems remain unresolved.
A significant problem with conventional flow cytometry technology may be that aerosols, containing hazardous materials, are generated around the nozzle during and after the sorting of the cells. One aspect of this problem may be that these hazardous, dangerous, or undesirable aerosols or contaminants can be adsorbed on the surfaces of the flow cytometer resulting in premature wear. Another aspect of this problem may be that these undesirable aerosols may be inhaled or absorbed through the skin of person(s) in proximity to the flow cytometer. Moreover, these aerosols may, with respect to samples or other chemicals, represent an undesired contaminant.
Another significant problem with conventional flow cytometry technology may be that the cells, biological materials associated with the cells, or other materials being analyzed with the flow cytometer may be susceptible to contamination, modification, molecular rearrangement, or the like, by exposure to the open air; constituents of the open air; or particles, chemicals, other biological materials, such as bacteria, viruses, pollen, microscopic flora or fauna, or other pathogens that can be carried by the open air.
A controlled sterile flow cytometer environment, for example, that prevents cells from being contaminated and also maintains their viability, may allow the sorted cells to be used for further processes such as genetic modification of the sorted cells themselves, isolation or preservation of the cells in a frozen state, or culturing of the isolated cells in conventional tissue culture media, administering the isolated cells to a patient, or various permutations and combinations of these or other processes. An example of such a combination might involve removing a sample of blood from a patient, labeling certain cells (e.g. pluripotent hematopoietic stem cells) with fluorescently labeled antibodies and isolating these cells using the sterile cell sorter by observing and measuring the emitted excitation wavelengths from these fluorescently labeled antibodies, culturing these isolated certain cells and genetically modifying them (e.g. using recombinant DNA technology) to provide genetically modified cells, and culturing these cells in a tissue culture media and injecting them back into the patient to provide a cure or remedy for a disease.
Another significant problem with conventional flow cytometer technology may be that protective enclosures presently manufactured such as those disclosed by U.S. Pat. Nos. 4,063,495; 4,045,192; 3,462,920; and 3,511,162, each hereby incorporated by reference, are arranged to substantially enclose the protected area and do not permit a great deal of access for manual operations, or other features that are useful with respect to flow cytometry procedures.
Another significant problem with conventional flow cytometer technology may be that the protective enclosures do not provide a disposable liner for the protective enclosure. As such, the protective enclosure itself can become contaminated with the cells, biological materials, or aerosoled chemicals, contaminants, or the like, which can then contaminate samples subsequently analyzed.
As to these problems associated with conventional flow cytometry technology, the instant invention addresses each with a practical solution.
Accordingly, a broad object of embodiments of the invention can be to provide an adjustably controllable environment containment system for a flow cytometer to protect both the particles being processed by the flow cytometer from contamination by both biological and non-biological materials and to protect persons using the flow cytometer from being exposed to the particles being processed and the chemistry utilized in the processing of such particles.
A significant object of embodiments of the invention can be to provide a environmental containment element (also referred to as an enclosure) that can separate the adjustably controlled environment within containment element from the undesired environment outside the containment element.
Another significant object of certain embodiments of the invention can be to provide an environmental containment element having a configuration such that upon introduction of a desired mixture of gases into such enclosure the fluid stream along the trajectory between orifice of the droplet generator to the collection container is maintained within such mixture of gases.
Another significant object of embodiments of the invention can be to protect the particles, or molecules embedded in the surface of such particles, being processed by a flow cytometer from reacting with the environment to produce unwanted modification(s) or molecular rearrangement(s); or being contaminated with microflora or microfauna, such as bacteria, viruses, pollen, or other biological pathogens, or the like.
Another significant object of embodiments of the invention can be to process particles (such as cells) in an environment that allows the products of the process to be used in further processes, such as genetic modification of the particles or cells themselves; allows isolation or preservation of the cells in a frozen state; culturing of the cells in tissue culture methods; or administering the processed or isolated cells, directly or after further processing into animal, including human, patients to provide a cure or remedy for disease.
Another significant object of embodiments of the invention can be to provide a method and devices for effectively cleaning the surface areas that surround the adjustably controlled environment.
Another significant object of embodiments of the invention can be to provide a flow cytometer that measures, but does not sort, cells. The ability to measure cells in a sterile environment is crucial when handling biological cells that are intended to be used for further cultivation or other processes requiring sterility.
Another significant object of embodiments of the invention can be to provide an enclosed environment that can be sterilized and furthermore, maintain its sterility during the actual measuring or sorting of cells. Although the present invention includes several subsystems, it is essential to have only certain subsystems located within the enclosed environment, but when these subsystems work in conjunction with those subsystems located outside the enclosed environment, the sterility of the enclosed environment can still be maintained.
Another significant object of embodiments of the invention can be to provide an enclosed environment that is functional. In other words, in addition to encompassing the subsystems that must be kept sterile, the enclosed environment must provide an environment in which the user, located outside the sterile environment can properly operate the cell sorter within the enclosed environment. The enclosed environment may include adequate work space for the operator, the proper equipment and tools as well as a storage place for them when not in use, a transfer port for transferring items in and out of the enclosed environment without breaking the sterility of the isolation chamber, adequate safety mechanisms to prevent the operator from being injured, and an interface for the operator to conveniently and comfortably work inside the sterile environment while located outside the enclosed environment.
Another significant object of embodiments of the invention can be to provide a method for sterilizing the enclosed environment before measuring and sorting the cells, and for maintaining a sterile air pressure within the enclosed environment during the measuring or sorting of the cells. Sterilization may be repeated at the end of measuring or sorting in order to decontaminate the enclosure when measuring or sorting cells or samples which may be infected (e.g. HIV infected blood). This sterilization after measuring or sorting protects the user of the cell sorter form unhealthy contaminants.
Another significant object of embodiments of the invention can be to provide a sterilization system to ensure that during the measuring and sorting of the cells, the isolation chamber is maintained at a pressure above atmospheric pressure which is used to maintain the sterility of the isolation chamber.
Naturally, further objects of the invention are disclosed through the description and by the drawings.